Poly-4-hydroxybutyrate (P4HB) and copolymers thereof can be produced using transgenic fermentation methods, see, for example, U.S. Pat. No. 6,548,569 to Williams et al., and are produced commercially, for example, by Tepha, Inc. (Lexington, Mass.). Poly-4-hydroxybutyrate (P4HB, TephaFLEX® biomaterial) is a strong, pliable thermoplastic polyester that, despite its biosynthetic route, has a relatively simple structure.

The polymer belongs to a larger class of materials called polyhydroxyalkanoates (PHAs) that are produced by numerous microorganisms (see, for example, Steinbüchel A., et al. Diversity of Bacterial Polyhydroxyalkanoic Acids, FEMS Microbial. Lett. 128:219-228 (1995)). In nature these polyesters are produced as storage granules inside cells, and serve to regulate energy metabolism. They are also of commercial interest because of their thermoplastic properties, and relative ease of production. Several biosynthetic routes are currently known to produce P4HB as shown in FIG. 1.
Chemical synthesis of P4HB has been attempted, but it has been impossible to produce the polymer with a sufficiently high molecular weight that is necessary for most applications (see Hori, Y., et al., Polymer 36:4703-4705 (1995); Houk, K. N., et al., J. Org. Chem., 2008, 73 (7), 2674-2678; and Moore, T., et al., Biomaterials 26:3771-3782 (2005)). In fact, it has been calculated to be thermodynamically impossible to chemically synthesize a high molecular weight homopolymer under normal conditions (Moore, T., et al., Biomaterials 26:3771-3782 (2005)).
U.S. Pat. Nos. 6,245,537, 6,623,748 and 7,244,442 describe methods of making PHAs with little to no endotoxin, which is suitable for medical applications. U.S. Pat. Nos. 6,548,569, 6,838,493, 6,867,247, 7,268,205, and 7,179,883 describe use of PHAs to make medical devices. Copolymers of P4HB include 4-hydroxybutyrate copolymerized with 3-hydroxybutyrate or glycolic acid (U.S. patent application No. 20030211131 by Martin and Skraly, U.S. Pat. No. 6,316,262 to Huisman et al., and U.S. Pat. No. 6,323,010 to Skraly et al.). Methods to control molecular weight of PHA polymers by biosynthetic methods have been disclosed by U.S. Pat. No. 5,811,272 to Snell et al.
PHAs with controlled degradation and degradation in vivo of less than one year are disclosed by U.S. Pat. Nos. 6,548,569, 6,610,764, 6,828,357, 6,867,248, and 6,878,758 to Williams et al. and WO 99/32536 to Martin et al. Applications of P4HB have been reviewed in Williams, S. F., et al., Polyesters, III, 4:91-127 (2002), and by Martin, D. et al. Medical Applications of Poly-4-hydroxybutyrate: A Strong Flexible Absorbable Biomaterial, Biochem. Eng. J. 16:97-105 (2003). Medical devices and applications of P4HB have also been disclosed by WO 00/56376 to Williams et al. Several patents including U.S. Pat. Nos. 6,555,123, 6,585,994, and 7,025,980 describe the use of PHAs in tissue repair and engineering.
In controlled release there currently exists a need for P4HB and copolymers thereof with narrowly defined weight average molecular weight ranges. By controlling these ranges, compositions can be developed that allow the controlled release of a wide range of bioactive agents. Particularly desired compositions include polymers with weight average molecular weights less than 250,000, and more preferably less than 100,000. Preferably, the compositions should have a polydispersity of less than 3, and more preferably less than 2.5.
In addition to being useful in controlled release, lower molecular weight compositions of P4HB and copolymers thereof can be useful in the preparation of other chemicals, polymers, copolymers, and for blending with other materials. For example, low molecular weight P4HB and copolymers thereof can be used in the preparation of polyurethanes or as nucleants or fillers in blends.
U.S. Pat. No. 6,623,730 to Williams and Martin discloses the use of oligomers of P4HB as prodrugs to modulate endogenous levels of 4-hydroxybutyrate, and methods to prepare these oligomers by hydrolysis of P4HB. The method disclosed uses aliquots of sodium methoxide to hydrolyze P4HB dissolved in a solvent (anhydrous THF) to provide the oligomers of P4HB of the desired molecular weight. Starting with a polymer of molecular weight 430,000 g/mol, sodium methoxide is used to produce P4HB molecular weights of 320,000, 82,000 and 25,000. The disadvantages of this procedure are as follows: (i) the use of methoxide can result in terminal esterification of the polyester chains (producing methyl esters); (ii) the relatively low solubility of P4HB in THF (and other solvents) requires large volumes of solvent to dissolve the polymer which is difficult to handle, expensive, and costly to remove; and (iii) basic conditions could potentially lead to elimination reactions in the polymer causing unwanted side reactions.
It is an object of the present invention to provide methods to produce P4HB and copolymers thereof by hydrolysis of high molecular weight P4HB and copolymers without the use of solvents.
It is a further object of the present invention to provide methods to produce P4HB and copolymers thereof by hydrolysis of high molecular weight P4HB and copolymers without the use of basic conditions that can esterify polymer terminal end groups or cause basic elimination in the polymer chains.
It is another object of the present invention to provide methods for hydrolysis of P4HB and copolymers thereof that maintain the polymers in solid state during hydrolysis resulting in easy isolation of the hydrolyzed products.
It is still another object of the present invention to provide compositions of P4HB and copolymers thereof that can be used in medical applications, for example, in implantable medical devices and or for the controlled release of bioactive substances, including the controlled release of 4-hydroxybutyrate.